PROJECT SUMMARY Interferon-inducible GTPases are among the most potent effectors in cell-autonomous immunity. They are dynamin-like large GTPases highly induced by proinflammatory cytokines, especially type I and II interferons. Interferon-inducible GTPases eliminate or restrict intracellular bacteria and protozoan parasites through a variety of strategies. The most prominent strategies are rupture of pathogen-containing vacuoles (PCVs) to expose pathogens to cytosolic pattern recognition receptors (PRRs), and direct attack and lysis of bacterial membranes. On the other hand, interferon-inducible GTPases are under tight control to avoid indiscriminate attack on host cell endomembranes. The critical roles of IFN-inducible GTPases are gaining attention and appreciation recently, yet the molecular mechanisms of their activation, their effector functions on target membranes, and their regulation by cellular and microbial factors remain evasive. In this proposal, we study the activation and regulation mechanisms of interferon-inducible GTPases using guanylate-binding protein 2 (GBP2) as the prototype. In our preliminary studies, we purified GBP2, determined its crystal structures, and gained initial insights into its structures and functions. In this research, we will take advantage of these preliminary data to determine the structural and mechanistic basis of GBP activation in solution and on target membrane, and the regulation mechanism by cellular and microbial factors. We will first elucidate GBP2 oligomerization status and GTPase activity in solution. We will then determine the atomic structures of GBP2 in various nucleotide-bound states using X-ray crystallography and cryogenic electron microscopy (cryo-EM). Next, we have modified GBP2 with farnesyl group and will characterize its interaction with lipids and liposomes. We will then determine the remodeling/lysis effect of farnesylated GBP2 on its target membranes using fluorescence-based liposome leakage assay and EM techniques. We also plan to characterize the higher-order assembly mode of membrane- attached GBP2. Finally, we will define how GBP2 is self-inhibited and regulated by other cellular and pathogen- derived proteins. Successful accomplishment of this proposed research will fill a major gap in our knowledge of the molecular mechanisms of intracellular pathogen detection and restriction. This work will also expand our understanding of activation and assembly of large GTPases in general. Ultimately, these findings will facilitate the development of novel therapeutic strategies for microbial infections and autoinflammatory diseases.